High quality ripe fruit results from a number of coordinated biochemical, metabolic, and storage changes which occur not only during ripening but also during fruit development prior to ripening. Collectively, these changes determine the final quantity and quality of the fruit. Some of the more commercially important traits include the import of sugars (principally sucrose) into the developing fruit from photosynthetically active portions of the plant and incorporation of this sugar into starch, accumulation of various organic acids in specific ratios, modifications of pigments involved in coloring of the fruit and changes in fungicidal or insecticidal compounds.
Polygalacturonase
In recent years Lycopersicon esculentum, the cultivated tomato, has become a popular system for studying fruit ripening. Tomato fruit ripening is characterized by a series of coordinated biochemical and physiological changes within the various subcellular compartments of the fruit tissue. These changes collectively contribute to the overall quality of the ripe fruit. The most obvious of the changes are alterations in fruit color, flavor, texture and resistance to certain pathogens.
One biochemical change in ripening fruit is the depolymerization and solubilization of cell wall polyuronides by the ripening-induced cell wall degrading enzyme, polygalacturonase (PG). PG activity increases dramatically during the ripening of many fruits, including tomato, and is the primary enzymic activity responsible for cell wall polyuronide degradation during fruit ripening. Reviewed in Giovannoni, et al, 1991 Ann. Rev. Hortic Sci: 67-103.
PG activity isolated from ripe tomato fruit is due to the presence of three structurally and immunologically-related isoforms of PG. These isoforms are termed PG1, PG2A and PG2B. (Ali, et al. Aust. J. Plant Physiol. 9:171, 1982). The PG2A and PG2B isoforms (45 and 46 kDa, respectively) appear well after the onset of ripening and are each composed of a single catalytic PG polypeptide differing only in degree of glycosylation. Because of the physical and biochemical similarity of PG2A and PG2B, the two isoforms shall be treated herein as a single isoform activity (the PG2 activity).
The PG1 isoform (approximately 100 kDa) is the first isoform to appear, at the onset of ripening, and is a heterodimer composed of the single catalytic PG2 polypeptide (either PG2A or PG2B) tightly associated with an ancillary cell wall glycoprotein, the PG .beta.-subunit. The formation of PG1 by association of the PG2 polypeptide with the PG .beta.-subunit protein alters both the biochemical and enzymic properties of the associated catalytic PG2 protein. The isoelectric point and pH optimum of PG1 are both a full unit lower than those of PG2. PG1 is more thermo-stable than PG2. PG1 retains complete activity after heating for 5 minutes at 65.degree. C., a treatment that completely inactivates PG2.
In recent years, CDNA clones for the catalytic PG2 polypeptide have been identified and used to examine in detail the regulation of PG gene expression in wild-type and mutant tomato fruit (DellaPenna, et al, Proc. Natl. Acad. Sci. USA 83:6420 (1986). Analysis of PG2 genomic and CDNA clones has revealed that the catalytic PG polypeptide is encoded by a single gene which is transcriptionally activated at the onset of wild-type fruit ripening (DellaPenna et al, Plant Physiol. 90:1372 (1989). PG2 mRNA is synthesized de novo during the ripening of wild-type fruit and accumulates to high levels, accounting for greater than 1% of the mRNA mass. Ripening-impaired mutants of tomato, which are inhibited in many ripening processes including PG2 expression, have greatly reduced levels of PG2 mRNA. The severe reduction in steady-state PG mRNA levels in the mutant genotypes is due to greatly reduced transcriptional activity of the PG gene (DellaPenna, et al, 1989, supra)
The PG .beta.-subunit has also been studied. The levels of PG .beta.-subunit increase approximately 4-fold during fruit ripening (Pressey, R., Eur. J. Biochem. 144:217-221 (1984)) and apparently determine the amount of PG1 produced during tomato ripening. Therefore, as PG .beta.-subunit levels are depleted (by formation of PG1), the timing of appearance of the PG2 isoform is also controlled.
While it is clear from in vitro studies that PG1 and PG2 differ in their biochemical properties, the physiological significance of the isoforms and the role of the PG .beta.-subunit protein remains uncertain. From a physiological point of view, it seems likely that a cell wall enzyme like PG might be localized or its activity restricted to specific regions of the cell wall by association with an adhesion or localizing factor, such as the PG .beta.-subunit protein. Recent results in transgenic systems have also suggested that PG1 may be the physiologically active isoform in vivo with regard to pectin degradation, presumably due to its association with the PG .beta.-subunit protein.
Pectolytic enzymes, such as PG, may have a role in plant pathogen interactions. Pathogen-derived pectolytic enzymes are thought to be important components of the mechanism by which pathogens penetrate and colonize plant tissues. Preliminary results from recently completed experiments have suggested that PG induction in transgenic mutant fruit increases colonization of the fruit by Alternaria alternata, a common late-season pathogen of wild-type tomato fruit to which mutant fruit are normally resistant. The apparent conferral of pathogen sensitivity to mutant fruit by the specific induction of PG expression suggests that increasing PG activity during fruit ripening may play an important role in altering the susceptibility of the fruit to pathogens.
Both classical breeding and genetic manipulations have been used to improve specific aspects of fruit quality. In recent years a number of genetic manipulations have been performed to modify specific ripening aspects of tomato fruit, often with commercially useful results. Such molecular approaches have definite advantages over classical breeding. The most obvious advantages are the accelerated development of genetically engineered varieties with enhanced traits, and the highly directed nature of the process. That is, specific biochemical steps carried out by proteins produced by single genes or small related gene families, can be targeted for modification by overexpressing the particular gene involved in the target step, thereby "enhancing or accelerating" the targeted cellular process. Conversely, by inhibiting the expression of a gene or gene family a biochemical process can be reduced or inhibited. Such approaches can very specifically modify a trait in an already existing, commercially useful plant without affecting other desired traits.
The expression of plant genes has been successfully modified by the following methods: overexpression of genes/proteins, antisense or cosuppression to inhibit gene expression, or transposon disruption of genes, which also inhibits expression of a particular gene. The first three methods (overexpression, antisense inhibition and cosuppression) rely on the directed expression of a specific gene construct at developmental and/or tissue specific stages of the plant's lifecycle where the process of interest is to be modified.
Gene expression is better altered by using developmental and tissue specific promoters to target and restrict the modifications of the process of interest to the tissues and/or developmental times in the plant lifecycle which will be least detrimental and most commercially beneficial to the overall growth and development of the plant. Promoters are DNA elements that direct the transcription of RNA in cells. Together with other regulatory elements that specify tissue and temporal specificity of gene expression, promoters control the development of organisms. However, this approach depends on the availability of a promoter specific enough to limit the expression of the transgene to the organ and developmental stage of interest, while simultaneously allowing expression at a high enough level to effectively modify the target gene in these tissues. What is needed is a promoter that will allow the targeting of chimeric gene expression to developing tomato fruit.